We are currently testing the hypothesis that non-cellular extracts from normal mammary tissue can reprogram non-mammary and cancer cell in the epithelium-divested mammary fat pad. Breast cancer is the second leading cause of cancer deaths in the United States. At present, the etiology of breast cancer is unknown; however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display 'normal' behavior when placed into embryonic (developmental) environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate and to prevent tumor formation. Pluripotent human embryonal carcinoma cells are redirected by the regenerating mammary gland microenvironment to contribute epithelial progeny for 'normal' gland development in-vivo. Human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation when mixed with mouse mammary epithelial cells. Immunochemistry for human-specific mitochondria, keratin 8 and 14, as well as human-specific milk proteins (alpha-lactalbumin in impregnated transplant hosts) confirmed the presence of human cell progeny. Features consistent with normal mammary gland development as seen in intact hosts (duct, lumen formation, development of secretory acini) were recapitulated in primary, secondary and tertiary outgrowths from chimeric implants. This suggests the dominance of the tissue microenvironment over tumorigenic cell fate. Experiments were conducted to redirect mouse Embryonic Stem (ES) cells from a tumorigenic phenotype to a normal mammary epithelial phenotype in vivo. Mixing LacZ-labeled ES cells with normal mouse mammary epithelial cells at ratios of 1:5 and 1:50 in phosphate buffered saline and immediately inoculating them into epithelium-divested mammary fat pads of immune-compromised mice accomplished this. Our results indicate that tumorigenesis occurs only when normal mammary ductal growth is not achieved in the inoculated fat pads. When normal mammary gland growth occurs, we find ES cells (LacZ+) progeny interspersed with normal mammary cell progeny in the mammary epithelial structures. These progeny, marked by LacZ expression, differentiate into multiple epithelial subtypes including steroid receptor positive luminal cells and smooth muscle actin-positive myoepithelial cells indicating that the ES cells are capable of epithelial multipotency in this context but do not form teratomas. In addition, in secondary transplants, ES cell progeny proliferate, contribute apparently normal mammary progeny, maintain their multipotency and do not produce teratomas. Our more recent studies indicate that ES cells are redirected to mammary epithelial cell fate when mixed with non-cellular extracellular matrix from rat mammary glands. The mouse mammary epithelial cell hierarchy encompasses both multipotent stem cells and those exhibiting lineage limited duct and lobular progenitor cell functions. An epithelial population distinct from the lobular progenitor is transformed during WAP-Int3 tumorigenesis. As expected, WAP-Int3/WAP-Cre/R26-beta-gal reporter (WAP-Int3/WC/R26) triple-transgenic mice failed to undergo secretory alveolar development, were unable to lactate and developed mammary tumors. Following pregnancy and involution, beta-gal-marked mammary epithelial cells were found in non-tumorigenic mammary tissue, but the resulting mammary tumors contained only beta-gal-negative cells. WAP-Int3/WC/R26 mammary glands contained ample ERa+ mammary epithelial cells (MEC), but only rare (1%) PR+ or RANKL+ cells. In addition, dissociated MEC from WAP-Int3/WC/R26 glands failed to regenerate a mammary tree upon transplantation into a cleared fat-pad of a nu/nu recipient mouse. However, when mixed with normal MEC, PI-MEC from WAP-Int3/WC/R26 mice contributed progeny to the resulting functional outgrowth. The WAP-Int3/WC/R26 derived PI-MEC displayed all of the properties of fully functional lobular progenitors including giving rise to ERa+, PR+, SMA+, and RANKL+ epithelial progeny. These results demonstrate that WAP-Int3 has no oncogenic effect upon lobular progenitors and that the expansion of functional lobular progenitors is required for secretory alveolar development and lactation. Furthermore, lobular progenitor function is ultimately controlled by signals within its microenvironment. We tested the hypothesis that paracrine signals from mammary epithelial cells expressing progesterone receptor (PR) or prolactin (Prl) are dispensable for redirection of testicular cells, and that re-directed wild-type testicular-derived mammary cells can rescue lobulogenesis of PR-null mammary epithelium by paracrine signaling during pregnancy. The testicular cells were redirected in vivo to mammary epithelial cell fate during regeneration of the mammary epithelium, and persisted in second-generation outgrowths. In the process, the redirected testicular cells rescued the developmentally deficient PR-null or Prl-null cells, signaling them through the paracrine factor RANKL to produce alveolar secretory structures during pregnancy. This is the first demonstration that paracrine signaling required for alveolar development is not required for cellular reprogramming in the mammary gland, and that reprogrammed testicular cells can provide paracrine signals to the surrounding mammary epithelium. Further experimentation showed that Amphiregulin (AR) null mammary epithelium redirected neural stem cells to a mammary epithelial cell fate despite its own poor growth in nulliparous or pregnant female hosts. Studies to determine the essential signals required from the mammary epithelium for redirection of non-mammary and cancer cells are ongoing.